The need for chronic immunosuppression represents a major limitation for islet transplantation in humans as a treatment or possible "cure" for type-1 diabetes. Therefore, inducing immune tolerance to islet transplants remains an important goal. Induction of mixed chimerism via allogeneic bone marrow transplantation currently represents a successful regimen for induction of immune tolerance in laboratory animals such as NOD mice, the best model for human type-1 diabetes. However, this procedure requires whole body irradiation (TBI) conditioning of the recipients. The toxicity of TBI-conditioning and the potential for graft versus host disease (GVHD) does not justify its use in the treatment of type-1 diabetes, and thus underscores the need for the development of successful radiation-free immune tolerance strategies. We have recently shown that, in the absence of irradiation, injection of high doses of donor bone marrow cells in combination with donor CD8+ T cells induces a mixed chimerism in prediabetic NOD mice preconditioned with anti-CD3 mAb. This allows for donor-specific tolerance and reversal of insulitis without induction of GVHD. The proposed studies will explore the mechanisms of GVHD prevention in this novel regimen. In our preliminary studies, anti-CD3-conditioned recipients showed low-level production of TNF-alpha and high-level production of IL-4 and IL-10 in the early period following donor CD8+ T cell injection. Therefore, we hypothesize that low-level production of Th1 cytokines and high-level production of Th2 cytokines in anti-CD3-conditioned recipients confines donor T cells to the host lymphohematopoietic system. Thus, donor CD8+ T cells facilitate donor stem cell engraftment in hematopoietic tissues without causing GVHD in epithelial tissues. We further hypothesize that the retention of donor T cells in host lymphohematopoietic tissues in anti-CD3-conditioned recipients is due to the lack of up-regulation of chemokine receptors including CCR5, CCR9, CCR10 and CXCR3 on donor CD8+ T cells; and the expression of chemokine receptors is reciprocally regulated by Th1 cytokines. The studies will 1) visualize donor CD8+ T cell migration and expansion in the lymphohematopoietic and epithelial tissues of NOD recipients conditioned with anti-CD3 as compared to recipients conditioned with TBI, using bioluminescence imaging; and determine whether or not donor CD8+ T cells in anti-CD3 conditioned recipients become anergic and/or apoptotic; 2) examine whether the expression pattern of chemokine receptors (i.e. CCR5, CCR9, CCR10 and CXCR3) of donor CD8+ T cells determine the trafficking pattern of donor CD8+ T cells in recipients conditioned with anti-CD3 or TBI; 3) examine whether or not production of IL-4 and IL-10 by host natural killer T (NKT) cells play a critical role in determining the trafficking pattern of donor CD8+ T and preventing GVHD in recipients conditioned with anti-CD3. These studies will provide new insights into mechanisms of GVHD prevention as well as promote the development of a radiation-free tolerance induction regimen applicable for islet cell transplantation and other organ transplantation in humans.